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170
Board Review Series Genetics
A
B
C
FIGURE 16-1. Karyotype chaos in a cancer cell. (A)
Photograph shows a normal human karyotype. (B) Photograph shows
an abnormal human karyotype due to a mutation involving the RAD 17 checkpoint protein, which plays a role in the cell
cycle. This mutation results in a re-replication of already replicated DNA and an abnormal karyotype. (C) Spectral kary-
otyping (24-color chromosome painting) shows twelve chromosome translocations (t) and two isochromosomes in a
human urinary bladder carcinoma. See Color Plate.
great majority of aneuploid cells undergo apoptosis, the few surviving cells will produce
progeny that are also aneuploid. The chromosomal aberrations get worse with each cell divi-
sion, eventually producing a cancer cell.
The phases of the cell cycle include:
G
0
(gap) phase, G
1
(gap) phase, S (synthesis) phase, G
2
(gap)
phase
, and the
M (mitosis) phase
(see Chapter 9).
III. CONTROL OF THE CELL CYCLE (Figure 16-2)
The control of the cell cycle involves three main components, which include:
A. Cdk-Cyclin Complexes.
The two main protein families that control the cell cycle are
cyclins
and the
cyclin-dependent protein kinases (Cdks).
A cyclin is a protein that regulates the activ-
ity of Cdks and is named because cyclins undergo a cycle of synthesis and degradation during
the cell cycle. The cyclins and Cdks form complexes called
Cdk-cyclin complexes
. The ability
of Cdks to phosphorylate target proteins is dependent on the particular cyclin that com-
plexes with it.
1. Cdk2-cyclin D and Cdk2-cyclin E
mediate the
G
1
S
S
S phase
transition at the
G
1
checkpoint.
2. Cdk1-cyclin A and Cdk1-cyclin B
mediate the
G
2
S
S
M phase
transition at the
G
2
checkpoint.
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Chapter 16
Genetics of Cancer
171
B. Checkpoints.
The checkpoints in the cell cycle are specialized, signaling mechanisms that
regulate and coordinate the cell response to
DNA damage
and
replication fork blockage
. When
the extent of DNA damage or replication fork blockage is beyond the steady-state threshold
of DNA repair pathways, a checkpoint signal is produced and a checkpoint is activated. The
activation of a checkpoint slows down the cell cycle so that DNA repair may occur and/or
blocked replication forks can be recovered.
This prevents DNA damage from being converted
into inheritable mutations producing highly transformed, metastatic cells
.
1. Control of the G
1
checkpoint.
There are three pathways that control the G
1
checkpoint
which include:
a.
Depending on the type of the DNA damage,
ATR kinase
and
ATM kinase
will activate
(i.e., phosphorylate)
Chk1 kinase
or
Chk2 kinase
, respectively. The activation of Chk1
kinase or Chk2 kinase causes the inactivation of
CDC25A phosphatase.
The inactivation
of CDC25A phosphatase causes the downstream stoppage at the G
1
checkpoint.
b.
Depending on the type of the DNA damage,
ATR kinase
and
ATM kinase
will activate
(i.e., phosphorylate)
p53
which allows p53 to disassociate from
Mdm2
. The activation of
p53 causes the transcriptional upregulation of
p21
. The binding of p21 to the Cdk2-
cyclin D and Cdk2-cyclin E inhibits their action and causes downstream stoppage at
the G
1
checkpoint.
c.
Depending on the type of the DNA damage,
ATR kinase
and
ATM kinase
will activate
(i.e., phosphorylate)
p16
which inactivates
Cdk4/6-cyclin D
and thereby causes down-
stream stoppage at the G
1
checkpoint.
2. Control of the G
2
checkpoint.
Depending on the type of the DNA damage,
ATR kinase
and
ATM
kinase
will activate (i.e., phosphorylate)
Chk1 kinase
or
Chk2 kinase
, respectively. The activa-
tion of Chk1 kinase or Chk2 kinase causes the inactivation of
CDC25C phosphatase
. The inac-
tivation of CDC25C phosphatase will cause the downstream stoppage at the G
2
checkpoint.
C. Inactivation of Cyclins.
Cyclins are inactivated by
protein degradation
during
anaphase of the
M phase
. The cyclin genes contain a homologous DNA sequence called a
destruction box.
A specific
recognition protein
binds to the amino acid sequence coded by the destruction
box, which allows
ubiquitin
(a 76 amino acid protein) to be covalently attached to lysine
residues of cyclin by the enzyme
ubiquitin ligase
. This process is called
polyubiquitination
.
Polyubiquitinated cyclins are rapidly degraded by proteolytic enzyme complexes called
pro-
teosomes
. Polyubiquitination is a widely occurring process for marking many different types
of proteins (cyclins are just a specific example) for rapid degradation.
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172
Board Review Series Genetics
DNA
damage
ssDNA
ATR
ATM
CDC25A
CDC25C
ChK2
ChK1
p53
p53
p21
E2F
E2F
G
1
checkpoint
G
0
G
1
(5 hrs)
G
2
(3 hrs)
M
(1 hr)
S
(7 hrs)
ProphasePrometaphaseMetaphaseAnaphase
T
elophase
Cytokinesis
G
2
checkpoint
+
+
RB
RB
cdk2-cyclin D
cdk2-cyclin E
cdk1-cyclin A
cdk1-cyclin B
cdk4/6-cyclin D
p16
Mdm2
Mdm2
DNA
damage
Double strand
DNA breaks
STOP
STOP
STOP
STOP
ChK2
ChK1
p16
PO4
PO4
PO4
PO4
PO4
FIGURE 16-2. Diagram of the cell cycle with checkpoints and signaling mechanisms.
ATR kinase responds to the sustained
presence of single-stranded DNA (ssDNA) because ssDNA is generated in virtually all types of DNA damage and replica-
tion fork blockage by activation (i.e., phosphorylation) of Chk1 kinase, p53, and p16. ATM kinase responds particularly to
double-stranded DNA breaks
by activation (i.e., phosphorylation) of Chk2 kinase, p53, and p16. The downstream pathway
past the STOP sign is as follows: Cdk2-cyclinD, Cdk2-cyclinE, and Cdk4/6-cyclinD phosphorylate the E2F-RB complex, which
causes phosphorylated RB to disassociate from E2F. E2F is a transcription factor that causes the expression of gene prod-
ucts that stimulate the cell cycle. Note the location of the four stop signs.
→
activation, inactivation.
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Chapter 16
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173
IV. PROTO-ONCOGENES AND ONCOGENES
A. Definitions.
1.
A
proto-oncogene
is a normal gene that encodes a protein involved in
stimulation of the cell
cycle
. Because the cell cycle can be regulated at many different points, proto-oncogenes
fall into many different classes (i.e.,
growth factors, receptors, signal transducers,
and
tran-
scription factors
).
2.
An
oncogene
is a mutated proto-oncogene that encodes for an
oncoprotein
involved in the
hyperstimulation of the cell cycle,
leading to oncogenesis. This is because the mutations
caused increased activity of the oncoprotein (either a hyperactive oncoprotein or
increased amounts of normal protein), not a loss of activity of the oncoprotein.
B. Alteration of a Proto-Oncogene to an Oncogene.
We know now that the vast majority of human
cancers are not caused by viruses. Instead, most human cancers are caused by the alteration
of proto-oncogenes so that oncogenes are formed producing an oncoprotein. The mecha-
nisms by which proto-Oncogenes are altered include:
1. Point mutation.
A point mutation (i.e., a
gain-of-function mutation
) of a proto-oncogene leads
to the formation of an oncogene. A
single mutant allele
is sufficient to change the phenotype
of a cell from normal to cancerous (i.e., a
dominant mutation
). This results in a hyperactive
oncoprotein that hyperstimulates the cell cycle leading to oncogenesis. Note: proto-onco-
genes only require a mutation in one allele for the cell to become oncogenic, whereas tumor
suppressor genes require a mutation in both alleles for the cell to become oncogenic.
2. Translocation
(see Chapter 11). A translocation results from breakage and exchange of seg-
ments between chromosomes. This may result in the formation of an oncogene (also
called a fusion gene or chimeric gene) which encodes for an oncoprotein (also called a
fusion protein or chimeric protein). A good example is seen in chronic myeloid leukemia
(CML). CML
t(9;22)(q34;q11)
is caused by a reciprocal translocation between chromosomes
9 and 22 with breakpoints at q34 and q11, respectively. The resulting der(22) is referred to
as the
Philadelphia chromosome
. This results in a hyperactive oncoprotein that hyperstim-
ulates the cell cycle leading to oncogenesis.
3. Amplification.
Cancer cells may contain hundreds of extra copies of proto-oncogenes.
These extra copies are found as either small paired chromatin bodies separated from the
chromosomes (double minutes) or as insertions within normal chromosomes. This
results in increased amounts of normal protein that hyperstimulates the cell cycle leading
to oncogenesis.
4. Translocation into a transcriptionally active region.
A translocation results from breakage
and exchange of segments between chromosomes. This may result in the formation of an
oncogene by placing a gene in a transcriptionally active region. A good example is seen in
Burkitt Lymphoma.
Burkitt Lymphoma t(8;14)(q24;q32)
is caused by a reciprocal transloca-
tion between band q24 on chromosome 8 and band q32 on chromosome 14. This results
in placing the
MYC gene
on chromosome 8q24 in close proximity to the
IGH gene
locus
(i.e., an immunoglobulin gene locus) on chromosome 14q32, thereby putting the MYC
gene in a transcriptionally active area in B lymphocytes (or antibody-producing plasma
cells). This results in increased amounts of normal protein that hyperstimulates the cell
cycle leading to oncogenesis.
D. Mechanism of Action of the RAS Gene: A Proto-Oncogene (Figure 16-3).
E. A List of Proto-Oncogenes (Table 16-1).
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A
tumor-suppressor gene
is a normal gene that encodes a protein involved in
suppression
of the cell cycle
. Many human cancers are caused by
loss-of-function mutations
of tumor-
suppressor genes. Note: tumor suppressor genes require a mutation in both alleles for a
cell to become oncogenic, whereas proto-oncogenes only require a mutation in one allele
for a cell to become oncogenic. Tumor suppressor genes can be either “gatekeepers” or
“caretakers”.
A. Gatekeeper Tumor Suppressor Genes.
These genes encode for proteins that either regulate the
transition of cells through the checkpoints (“gates”) of the cell cycle or promote apoptosis.
This prevents oncogenesis. Loss-of-function mutations in gatekeeper tumor-suppressor
genes lead to oncogenesis.
B. Caretaker Tumor Suppressor Genes.
These genes encode for proteins that either detect/repair
DNA mutations or promote normal chromosomal disjunction during mitosis. This prevents
oncogenesis by maintaining the integrity of the genome. Loss-of-function mutations in care-
taker tumor suppressor genes lead to oncogenesis.
C. Mechanism of Action of the RB1 Gene: A Tumor-Suppressor Gene (Retinoblastoma; Figure 16-4).
D. Mechanism of Action of the TP53 Gene: A Tumor-Suppressor Gene (“Guardian of the Genome”;
Figure 16-5).
E. A List of Tumor-Suppressor Genes (Table 16-2).
VI. HEREDITARY CANCER SYNDROMES
A. Hereditary Retinoblastoma (RB).
1.
Hereditary RB is an
autosomal dominant
genetic disorder caused by a mutation in the
RB1
gene
on
chromosome 13q14.1-q14.2
for the
retinoblastoma (RB) associated protein (p110
RB
)
.
1,000 different mutations of the RB1 gene have been identified which include missense,
frameshift, and RNA splicing mutations, which result in a premature STOP codon, and a
loss-of-function mutation
.
2.
RB protein binds to
E2F
(a gene regulatory protein) such that there will be no expression of
target genes whose gene products stimulate the cell cycle at the G1 checkpoint. The RB
protein belongs to the family of
tumor-suppressor genes
.
3.
Hereditary RB affected individuals inherit one mutant copy of the RB1 gene from their
parents (an inherited germline mutation) followed by a somatic mutation of the second
copy of the RB1 gene later in life.
4. Parents of the proband.
The proband may have a RB affected parent or an unaffected par-
ent who has an RB1 gene mutation. If the proband mutation is identified in either parent,
then the parent is at risk of transmitting that RB1 gene mutation to other offspring. If the
proband mutation is not identified in either parent, then the proband has a de novo RB1
gene germline mutation (90% to 94% chance) or one parent is mosaic for the RB1 gene
mutation (6% to 10% chance).
5. Siblings of the proband.
The risk to each sibling of the proband of inheriting the RB1 gene
germline mutation is 50% if a parent has the same RB1 gene germline mutation identified
in the proband. The risk to each sibling of the proband is 3% (due to the possibility of
germline mosaicism in one parent) if neither parent has the same RB1 gene germline
mutation identified in the proband.
6. Offspring of the proband.
The risk to the offspring of the proband is 50% if the proband has
hereditary RB (bilateral tumors).
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